Breast cancer cells commonly metastasize to the brain using mechanisms that are not fully understood. Because there are no effective therapeutics and surgical treatments are often problematic, the survival rate of breast cancer patients that develop brain metastases is dismal (<10 months). Recent work indicates that only metastatic breast cancer cells that attach to the vascular surface in the brain survive to form microtumors. Cells that invade deep into the brain parenchyma do not survive. These findings indicate the brain microvasculature provides a unique niche for breast cancer cell survival and propagation. However, it is not known how breast cancer cells attach to the vessel wall and communicate with the endothelium. Our recent published findings and preliminary results presented in this proposal demonstrate that Cx43-mediated gap junction (GJ) adhesion and communication with the brain vasculature is required for brain metastasis. Furthermore, we find that brain homing, metastatic cells, represent a subpopulation of tumor cells with stem cell-like properties (referred to as breast cancer stem cells, BCSCs). Importantly, BCSCs specifically express Cx43, which facilitates robust formation of Cx43-GJs with the Cx43-enriched brain vasculature. These findings are important because it provides a plausible explanation for why breast cancer cells commonly home to the brain and grow in association with the brain vasculature. Our findings also provide a unique mechanism to target metastatic cells in the brain with established GJ therapeutics such as carbenoxolone (CBX) and function blocking, cell-permeable peptides, which are currently being evaluated in clinical trials. Therefore, work i this proposal will determine precisely how heterocellular GJ communication and signal transduction regulates BCSC olonization of the brain vasculature. For these studies, we will use powerful fluorescent stem cell reporter constructs and specific gain or loss of function cx43 gene mutants in combination with unique preclinical animal models that facilitate intravital tracking and analysis of BCSC metastasis in the brain. Our work will provide a fundamental understanding of how BCSCs colonize the brain using GJ communication and determine if perturbation of this communication network can be used to treat metastatic disease in the brain.